An important question in neurobiology is how portions of the undifferentiated neural tube become regionally specified. Although much is now known about the genes involved in specification and patterning the spinal cord and hindbrain of vertebrates, surprisingly little information exists on the genes involved in specification and patterning of the vertebrate anterior brain. Two zebrafish homeodomain genes, zOtx1 and zOtx2, which begin to be expressed during mid-gastrula in a sharply defined region that probably corresponds at this stage to the prospective forebrain and midbrain, have been isolated in the investigator's laboratory. These two zOtx genes will be utilized to investigate how the forebrain and midbrain is formed in the zebrafish embryo. These investigations integrate fate mapping, tests of cell commitment, and molecular genetic analysis, to attempt an understanding of the early events of forebrain and midbrain patterning. The specific aims of the proposal are as follows: A) To determine the fate map of the anterior brain of the zebrafish embryo. These experiments will be done by injecting fluorescein-dextran into individual gastrula cells and by the release of fluorescein from a caged complex in a discrete region of the gastrula. B) To determine the timing of commitment of cells to a forebrain or midbrain fate, by utilizing cell transplantation techniques. The time at which prospective forebrain and midbrain cells are committed to express markers characteristic of these neural regions will be tested by transplanting cells of increasing age to non-neural sites in early gastrula embryos and to embryos of the same ae as the donor cells. Also to be tested will be the degree to which non-neural cells can respond to the signals involved in the establishment of anterior brain identify. C) To test for function of the Otx genes in forebrain and midbrain patterning, by investigating the effects of ectopic expression and overexpression of the gene products, and by utilizing a number of approaches to obtain a loss of function of the gene products. This project should increase our knowledge of the early events in specification of the forebrain and midbrain. The studies of may yield information relevant to the holoprosencephalies, a major class of inherited syndromes in which parts or the anterior brain are lacking.